Visualization of alveolar recruitment in a porcine model of unilateral lung lavage using 3He-MRI

BACKGROUND: In the acute respiratory distress syndrome potentially recruitable lung volume is currently discussed. (3)He-magnetic resonance imaging ((3)He-MRI) offers the possibility to visualize alveolar recruitment directly. METHODS: With the approval of the state animal care committee, unilateral lung damage was induced in seven anesthetized pigs by saline lavage of the right lungs. The left lung served as an intraindividual control (healthy lung). Unilateral lung damage was confirmed by conventional proton MRI and spiral-CT scanning. The total aerated lung volume was determined both at a positive end-expiratory pressure (PEEP) of 0 and 10 mbar from three-dimensionally reconstructed (3)He images, both for healthy and damaged lungs. The fractional increase of aerated volume in damaged and healthy lungs, followed by a PEEP increase from 0 to 10 mbar, was compared. RESULTS: Aerated gas space was visualized with a high spatial resolution in the three-dimensionally reconstructed (3)He-MR images, and aeration defects in the lavaged lung matched the regional distribution of atelectasis in proton MRI. After recruitment and PEEP increase, the aerated volume increased significantly both in healthy lungs from 415 ml [270-445] (median [min-max]) to 481 ml [347-523] and in lavaged lungs from 264 ml [71-424] to 424 ml [129-520]. The fractional increase in lavaged lungs was significantly larger than that in healthy lungs (healthy: 17% [11-38] vs. lavage: 42% [14-90] (P=0.031). CONCLUSION: The (3)He-MRI signal might offer an experimental approach to discriminate atelectatic vs. poor aerated lung areas in a lung damage animal model. Our results confirm the presence of potential recruitable lung volume by either alveolar collapse or alveolar flooding, in accordance with previous reports by computed tomography.

Characterization of the 9L gliosarcoma implanted in the Fischer rat: an orthotopic model for a grade IV brain tumor.

Among rodent models for brain tumors, the 9L gliosarcoma is one of the most widely used. Our 9L-European Synchrotron Radiation Facility (ESRF) model was developed from cells acquired at the Brookhaven National Laboratory (NY, USA) in 1997 and implanted in the right caudate nucleus of syngeneic Fisher rats. It has been largely used by the user community of the ESRF during the last decade, for imaging, radiotherapy, and chemotherapy, including innovative treatments based on particular irradiation techniques and/or use of new drugs. This work presents a detailed study of its characteristics, assessed by magnetic resonance imaging (MRI), histology, immunohistochemistry, and cytogenetic analysis. The data used for this work were from rats sampled in six experiments carried out over a 3-year period in our lab (total number of rats = 142). The 9L-ESRF tumors were induced by a stereotactic inoculation of 10(4) 9L cells in the right caudate nucleus of the brain. The assessment of vascular parameters was performed by MRI (blood volume fraction and vascular size index) and by immunostaining of vessels (rat endothelial cell antigen-1 and type IV collagen). Immunohistochemistry and regular histology were used to describe features such as tumor cell infiltration, necrosis area, nuclear pleomorphism, cellularity, mitotic characteristics, leukocytic infiltration, proliferation, and inflammation. Moreover, for each of the six experiments, the survival of the animals was assessed and related to the tumor growth observed by MRI or histology. Additionally, the cytogenetic status of the 9L cells used at ESRF lab was investigated by comparative genomics hybridization analysis. Finally, the response of the 9L-ESRF tumor to radiotherapy was estimated by plotting the survival curves after irradiation. The median survival time of 9L-ESRF tumor-bearing rats was highly reproducible (19-20 days). The 9L-ESRF tumors presented a quasi-exponential growth, were highly vascularized with a high cellular density and a high proliferative index, accompanied by signs of inflammatory responses. We also report an infiltrative pattern which is poorly observed on conventional 9 L tumor. The 9L-ESRF cells presented some cytogenetic specificities such as altered regions including CDK4, CDKN2A, CDKN2B, and MDM2 genes. Finally, the lifespan of 9L-ESRF tumor-bearing rats was enhanced up to 28, 35, and 45 days for single doses of 10, 20, and 2 × 20 Gy, respectively. First, this report describes an animal model that is used worldwide. Second, we describe few features typical of our model if compared to other 9L models worldwide. Altogether, the 9L-ESRF tumor model presents characteristics close to the human high-grade gliomas such as high proliferative capability, high vascularization and a high infiltrative pattern. Its response to radiotherapy demonstrates its potential as a tool for innovative radiotherapy protocols.

Histomorphometric and Radiographical Changes After Lumbar Implantation of the PEEK Nonfusion Interspinous Device in the BB.4S Rat Model

Study Design. An experimental animal study. Objective. To investigate histomorphometric and radiographical changes in the BB.4S rat model after PEEK (polyetheretherketone) nonfusion interspinous device implantation. Summary of Background Data. Clinical effectiveness of the PEEK nonfusion spine implant Wallis (Abbott, Bordeaux, France; now Zimmer, Warsaw, IN) is well documented. However, there is a lack of evidence on the long-term effects of this implant on bone, in particular its influence on structural changes of bone elements of the lumbar spine. Methods. Twenty-four male BB.4S rats aged 11 weeks underwent surgery for implantation of a PEEK nonfusion interspinous device or for a sham procedure in 3 groups of 8 animals each: 1) implantation at level L4–L5; 2) implantation at level L5–L6; and 3) sham surgery. Eleven weeks postoperatively osteolyses at the implant-bone interface were measured via radiograph, bone mineral density of vertebral bodies was analyzed using osteodensitometry, and bone mineral content as well as resorption of the spinous processes were examined by histomorphometry. Results. Resorption of the spinous processes at the site of the interspinous implant was found in all treated segments. There was no significant difference in either bone density of vertebral bodies or histomorphometric structure of the spinous processes between adjacent vertebral bodies, between treated and untreated segments and between groups. Conclusion. These findings indicate that resorption of spinous processes because of a result of implant loosening, inhibit the targeted load redistribution through the PEEK nonfusion interspinous device in the lumbar spinal segment of the rat. This leads to reduced long-term stability of the implant in the animal model. These results suggest that PEEK nonfusion interspinous devices like the Wallis implants may have time-limited effects and should only be used for specified indications.

Levels of matrix metalloproteinase-9 within cerebrospinal fluid in a rabbit model of coccidioidal meningitis and vasculitis.

Matrix metalloproteinase (MMP)-9 is produced by the central nervous system and inflammatory cells in a variety of inflammatory conditions in both animals and humans. MMP-9 promotes inflammation, breakdown of the blood-brain barrier, and vasculitis. Because vasculitis is seen frequently in patients with coccidioidal meningitis (CM), this study evaluated the presence of MMP-9 within the cerebrospinal fluid (CSF) of rabbits infected intracisternally with Coccidioides immitis arthroconidia. Infected rabbits demonstrated systemic and neurological sequelae to infection, including CSF pleocytosis. Levels of MMP-9 within CSF were assayed by use of zymography and compared with MMP-2 levels, which served as an internal control. Elevated levels of MMP-9 were detectable by day 3, continued to increase through day 10, and declined by day 15 after infection. MMP-9 may contribute to inflammation and vasculitis in this animal model. Future work can focus on evaluation of MMP inhibitors, to gain a better perspective of the role of this MMP in CM.

Dominance of chemokine ligand 2 and matrix metalloproteinase-2 and -9 and suppression of pro-inflammatory cytokines in the epidural compartment after intervertebral disc extrusion in a canine model

BACKGROUND CONTEXT In canine intervertebral disc (IVD) disease, a useful animal model, only little is known about the inflammatory response in the epidural space. PURPOSE To determine messenger RNA (mRNA) expressions of selected cytokines, chemokines, and matrix metalloproteinases (MMPs) qualitatively and semiquantitatively over the course of the disease and to correlate results to neurologic status and outcome. STUDY DESIGN/SETTING Prospective study using extruded IVD material of dogs with thoracolumbar IVD extrusion. PATIENT SAMPLE Seventy affected and 13 control (24 samples) dogs. OUTCOME MEASURES Duration of neurologic signs, pretreatment, neurologic grade, severity of pain, and outcome were recorded. After diagnostic imaging, decompressive surgery was performed. METHODS Messenger RNA expressions of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF), interferon (IFN)γ, MMP-2, MMP-9, chemokine ligand (CCL)2, CCL3, and three housekeeping genes was determined in the collected epidural material by Panomics 2.0 QuantiGene Plex technology. Relative mRNA expression and fold changes were calculated. Relative mRNA expression was correlated statistically to clinical parameters. RESULTS Fold changes of TNF, IL-1β, IL-2, IL-4, IL-6, IL-10, IFNγ, and CCL3 were clearly downregulated in all stages of the disease. MMP-9 was downregulated in the acute stage and upregulated in the subacute and chronic phase. Interleukin-8 was upregulated in acute cases. MMP-2 showed mild and CCL2 strong upregulation over the whole course of the disease. In dogs with severe pain, CCL3 and IFNγ were significantly higher compared with dogs without pain (p=.017/.020). Dogs pretreated with nonsteroidal anti-inflammatory drugs revealed significantly lower mRNA expression of IL-8 (p=.017). CONCLUSIONS The high CCL2 levels and upregulated MMPs combined with downregulated T-cell cytokines and suppressed pro-inflammatory genes in extruded canine disc material indicate that the epidural reaction is dominated by infiltrating monocytes differentiating into macrophages with tissue remodeling functions. These results will help to understand the pathogenic processes representing the basis for novel therapeutic approaches. The canine IVD disease model will be rewarding in this process.

Effect of enamel matrix derivative on periodontal wound healing and regeneration in an osteoporotic model.

BACKGROUND Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis-related periodontal defects. METHODS Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD-treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD-treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro-CT) analysis. The number of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. RESULTS OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by micro-CT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165μm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP-positive osteoclasts did not vary between untreated and EMD-treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMD-treated samples. CONCLUSIONS The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis.

The Silencing of N-myc Downstream-Regulated Gene-1 in an Orthotopic Pancreatic Cancer Model Leads to More Aggressive Tumor Growth and Metastases

BACKGROUND: The understanding of molecular mechanisms leading to poor prognosis in pancreatic cancer may help develop treatment options. N-myc downstream-regulated gene-1 (NDRG1) has been correlated to better prognosis in pancreatic cancer. Therefore, we thought to analyze how the loss of NDRG1 affects progression in an orthotopic xenograft animal model of recurrence. METHODS: Capan-1 cells were silenced for NDRG1 (C(sil)) or transfected with scrambled shRNA (C(scr)) and compared for anchorage-dependent and anchorage-independent growth, invasion and tube formation in vitro. In an orthotopic xenograft model of recurrence tumors were grown in the pancreatic tail. The effect of NDRG1 silencing was evaluated on tumor size and metastasis. RESULTS: The silencing of NDRG1 in Capan-1 cells leads to more aggressive tumor growth and metastasis. We found faster cell growth, double count of invaded cells and 1.8-fold increase in tube formation in vitro. In vivo local tumors were 5.9-fold larger (p = 0.006) and the number of metastases was higher in animals with tumors silenced for NDRG1 primarily (3 vs. 1.1; p = 0.005) and at recurrence (3.3 vs. 0.9; p = 0.015). CONCLUSION: NDRG1 may be an interesting therapeutic target as its silencing in human pancreatic cancer cells leads to a phenotype with more aggressive tumor growth and metastasis.

The impact of environmental enrichment on the outcome variability and scientific validity of laboratory animal studies.

It has been widely accepted for some time that species-appropriate environmental enrichment is important for the welfare of research animals, but its impact on research data initially received little attention. This has now changed, as the use of enrichment as one element of routine husbandry has expanded. In addition to its use in the care of larger research animals, such as nonhuman primates, it is now being used to improve the environments of small research animals, such as rodents, which are used in significantly greater numbers and in a wide variety of studies. Concern has been expressed that enrichment negatively affects both experimental validity and reproducibility. However, when a concise definition of enrichment is used, with a sound understanding of the biology and behaviour of the animal as well as the research constraints, it becomes clear that the welfare of research animals can be enhanced through environmental enrichment without compromising their purpose. Indeed, it is shown that the converse is true: the provision of suitable enrichment enhances the well-being of the animal, thereby refining the animal model and improving the research data. Thus, the argument is made that both the validity and reproducibility of the research are enhanced when proper consideration is given to the research animal's living environment and the animal's opportunities to express species-typical behaviours.

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model.

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

Un modèle animal simple pour l'apprentissage des techniques de microanastomoses vasculaires de congruences diefférentes

BACKGROUND Since the pioneering work of Jacobson and Suarez, microsurgery has steadily progressed and is now used in all surgical specialities, particularly in plastic surgery. Before performing clinical procedures it is necessary to learn the basic techniques in the laboratory. OBJECTIVE To assess an animal model, thereby circumventing the following issues: ethical rules, cost, anesthesia and training time. METHODS Between July 2012 and September 2012, 182 earthworms were used for 150 microsurgical trainings to simulate discrepancy microanastomoses. Training was undertaken over 10 weekly periods. Each training session included 15 simulations of microanastomoses performed using the Harashina technique (earthworm diameters >1.5 mm [n=5], between 1.0 mm and 1.5 mm [n=5], and <1.0 mm [n=5]). The technique is presented and documented. A linear model with main variable as the number of the week (as a numeric covariate) and the size of the animal (as a factor) was used to determine the trend in time of anastomosis over subsequent weeks as well as differences between the different size groups. RESULTS The linear model showed a significant trend (P<0.001) in time of anastomosis in the course of the training, as well as significant differences (P<0.001) between the groups of animal of different sizes. For diameter >1.5 mm, mean anastomosis time decreased from 19.6±1.9 min to 12.6±0.7 min between the first and last week of training. For training involving smaller diameters, the results showed a reduction in execution time of 36.1% (P<0.01) (diameter between 1.0 mm and 1.5 mm) and 40.6% (P<0.01) (diameter <1.0 mm) between the first and last weeks. The study demonstrates an improvement in the dexterity and speed of nodes' execution. CONCLUSION The earthworm appears to be a reliable experimental model for microsurgical training of discrepancy microanastomoses. Its numerous advantages, as discussed in the present report, show that this model of training will significantly grow and develop in the near future.

Pre-clinical in vivo models for the screening of bone biomaterials for oral/craniofacial indications: focus on small-animal models.

Preclinical in vivo experimental studies are performed for evaluating proof-of-principle concepts, safety and possible unwanted reactions of candidate bone biomaterials before proceeding to clinical testing. Specifically, models involving small animals have been developed for screening bone biomaterials for their potential to enhance bone formation. No single model can completely recreate the anatomic, physiologic, biomechanic and functional environment of the human mouth and jaws. Relevant aspects regarding physiology, anatomy, dimensions and handling are discussed in this paper to elucidate the advantages and disadvantages of small-animal models. Model selection should be based not on the 'expertise' or capacities of the team, but rather on a scientifically solid rationale, and the animal model selected should reflect the question for which an answer is sought. The rationale for using heterotopic or orthotopic testing sites, and intraosseous, periosseous or extraskeletal defect models, is discussed. The paper also discusses the relevance of critical size defect modeling, with focus on calvarial defects in rodents. In addition, the rabbit sinus model and the capsule model in the rat mandible are presented and discussed in detail. All animal experiments should be designed with care and include sample-size and study-power calculations, thus allowing generation of meaningful data. Moreover, animal experiments are subject to ethical approval by the relevant authority. All procedures and the postoperative handling and care, including postoperative analgesics, should follow best practice.

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury

BACKGROUND CONTEXT In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

Waddlia chondrophila induces systemic infection, organ pathology, and elicits Th1-associated humoral immunity in a murine model of genital infection

Waddlia chondrophila is a known bovine abortigenic Chlamydia-related bacterium that has been associated with adverse pregnancy outcomes in human. However, there is a lack of knowledge regarding how W. chondrophila infection spreads, its ability to elicit an immune response and induce pathology. A murine model of genital infection was developed to investigate the pathogenicity and immune response associated with a W. chondrophila infection. Genital inoculation of the bacterial agent resulted in a dose-dependent infection that spread to lumbar lymph nodes and successively to spleen and liver. Bacterial-induced pathology peaked on day 14, characterized by leukocyte infiltration (uterine horn, liver, and spleen), necrosis (liver) and extramedullary hematopoiesis (spleen). Immunohistochemistry demonstrated the presence of a large number of W. chondrophila in the spleen on day 14. Robust IgG titers were detected by day 14 and remained high until day 52. IgG isotypes consisted of high IgG2a, moderate IgG3 and no detectable IgG1, indicating a Th1-associated immune response. This study provides the first evidence that W. chondrophila genital infection is capable of inducing a systemic infection that spreads to major organs, induces uterus, spleen, and liver pathology and elicits a Th1-skewed humoral response. This new animal model will help our understanding of the mechanisms related to intracellular bacteria-induced miscarriages, the most frequent complication of pregnancy that affects one in four women.

Chimpanzee: A predictive model for the human cytochrome P450 3A subfamily

The human cytochrome P450 3A (CYP3A) subfamily is responsible for most of the metabolism of therapeutic drugs; however, an adequate in vivo model has yet to be discovered. This study begins with an investigation of a controversial topic surrounding the human CYP3As--estrogen regulation. A novel approach to this topic was used by defining expression in the estrogen-responsive endometrium. This study shows that estrogen down-regulates CYP3A4 expression in the endometrium. On the other hand, analogous studies showed an increase in CYP3A expression as age increases in liver tissue. Following the discussion of estrogen regulation, is an investigation of the cross-species relationships among all of the CYP3As was completed. The study compares isoforms from piscines, avians, rodents, canines, ovines, bovines, and primates. Using the traditional phylogenetic analyses and employing a novel approach using exon and intron lengths, the results show that only another primate could be the best animal model for analysis of the regulation of the expression of the human CYP3As. This analysis also demonstrated that the chimpanzee seems to be the best available human model. Moreover, the study showed the presence and similarities of one additional isoform in the chimpanzee genome that is absent in humans. Based on these results, initial characterization of the chimpanzee CYP3A subfamily was begun. While the human genome contains four isoforms--CYP3A4, CYP3A5, CYP3A7, and CYP3A43--the chimpanzee genome has five, the four previously mentioned and CYP3A67. Both species express CYP3A4, CYP3A5, and CYP3A43, but humans express CYP3A7 while chimpanzees express CYP3A67. In humans, CYP3A4 is expressed at higher levels than the other isoforms, but some chimpanzee individuals express CYP3A67 at higher levels than CYP3A4. Such a difference is expected to alter significantly the total CYP3A metabolism. On the other hand, any study considering individual isoforms would still constitute a valid method of study for the human CYP3A4, CYP3A5, and CYP3A43 isoforms. ^

A factor IX-deficient mouse model for hemophilia B gene therapy

We have generated a mouse where the clotting factor IX (FIX) gene has been disrupted by homologous recombination. The FIX nullizygous (−/−) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (−/−) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.